In a recent publication, the replication fidelity of HIV-1 reverse transcriptase (RT) was investigated under physiological conditions. It is well known that HIV-1 RT is error prone. This, in combination with the high replication rate of the virus, result in the frequent generation of mutant virions of which many are resistant to drugs used in antiretroviral treatment.
The low fidelity of HIV-1 RT is not unique to HIV-1 since RT enzymes from other organisms share this characteristic (e.g., Alfalfa mosaic virus and Murine Leukemia Virus RTs). However, in vitro research into HIV-1 RT enzymology has given the impression that this enzyme has a particularly low fidelity.
In a new publication by Achuthan and colleagues, the authors indicate that the low fidelity of HIV-1 RT is on par with that of most other RT enzymes, under physiological conditions. The amount of dNTP and free Mg2+ can both impact on the fidelity of the enzyme. In assays optimized for HIV-1 RT enzymatic activity, Mg2+ concentrations are usually well above physiologically levels (e.g. in lymphocytes). The authors show that low free Mg2+ levels lead to an increase in HIV RT fidelity, with an error frequency that is ~10 times lower.Therefore, although HIV-1 RT has a low fidelity it is essential that in vitro experiments, particularly those concerned with fidelity, are carried out at free Mg2+ concentrations that resemble those in the cellular environment.
Figure 1: PCR-based lacZ_ complementation system used to determine the fidelity of HIV RT. (A) An overview of the procedure used to assess polymerase fidelity. RNAs are indicated by broken lines and DNAs by solid lines. Primers have arrowheads at the 3= end. The _760-nucleotide template RNA used as the initial template for HIV RT RNA-directed DNA synthesis is shown at the top with the 3= and 5= ends indicated. The positions of PvuII and EcoRI restriction sites are indicated for reference to the vector. The filled box at the bottom of is the 115-base region of the lacZ_ gene that was scored in the assay. (B) Plasmid pBSM13PvuII1146 is shown. Relevant sites on the plasmid are indicated; numbering is based on that for the parent plasmid (pBSM13_ [Stratagene]). (C) The nucleotide and amino acid sequences for the 115-base region of the lacZ_ gene that was scored in the assay are shown. Both strands of the DNA plasmid are shown since HIV RT synthesis was performed in both directions (see panel A).
Human Immunodeficiency Virus Reverse Transcriptase Displays DramaticallyHigher Fidelity under Physiological Magnesium Conditions In Vitro
Vasudevan Achuthan, Brian J. Keith, Bernard A. Connolly, and Jeffrey J.DeStefano
J. Virol. August 2014 88:8514-8527; published ahead of print 21 May 2014,